Primer set, reagent kit and method for fluorescence detection of African swine fever virus through LAMP (loop-mediated isothermal amplification)
A technology for detection of African swine fever virus and fluorescence, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of easy contamination of detection reagent solutions and low sensitivity, and achieve reduced sample processing time and high efficiency. Sensitivity, low cost effect
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Embodiment 1
[0034] Design and verification of embodiment 1LAMP primers
[0035] 1. Design of primers
[0036] The characteristic of LAMP is that four primers are designed for six regions on the target gene, and the amplification reaction is carried out under constant temperature conditions by using a strand displacement DNA polymerase. The reaction can produce a large amount of amplification products, which can be combined with the fluorescent dye SYTO 9, which can be A fluorescence detection instrument detects the fluorescence signal. According to the ASFV Georgia strain published by Genbank (GenBank: MH910495.1), using the online biological software (http: / / primerexplorer.jp / ), by adjusting the Tm value, GC content, dG critical value, amplification length and fragment area, etc. Parameter values are designed to be suitable for LAMP-specific outer primers and inner primers. In the present invention, in order to improve the amplification efficiency, 1 loop primer is designed according ...
Embodiment 2
[0045] Example 2 The optimization of the annealing temperature of the LAMP method for detecting African swine fever virus on an isothermal fluorescent PCR amplification instrument
[0046] 1. Select the primer combination as: primer combination A in Table 1 of Example 1.
[0047] 2. The LAMP amplification reaction system is as in Example 1, that is, 13 μL of AFSV fluorescence reaction solution (A), 9 μL of AFSV fluorescence reaction solution (B), 1 μL of enzyme reaction solution, and 2 μL of sample nucleic acid.
[0048] 3. Carry out amplification on a loop-mediated isothermal amplification instrument, and set four temperatures of 58°C, 60°C, 63°C, and 65°C.
[0049] 4. Analysis results: the results show that the amplification effect is the best at 63°C, and 63°C is selected as the amplification temperature of the primer set A of the present invention.
Embodiment 3
[0050] Embodiment 3 sample processing
[0051] 1. Sample collection: Pigs that died or were culled, took lungs, tonsils and brain tissue; live pigs to be inspected, took 5mL of blood with a syringe, and dipped a cotton swab into a 1.5mL centrifuge tube filled with PBS. The collected samples should be stored at 2-8°C and sent to the laboratory for testing (it is required that the disease materials submitted for inspection should be fresh, and repeated freezing and thawing of disease materials is strictly prohibited).
[0052] 2. Sample processing: each sample is processed separately.
[0053] (1) Add 10 μL of the sample (whole blood, tissue, swab supernatant) to a 1.5 mL centrifuge tube, add 100 μl DNAExtract Solution A, vortex for 20 seconds, let stand at room temperature for 3-5 minutes (or incubate in a metal bath at 95°C for 3 -5min for better extraction). Wherein, DNA Extract Solution A contains 2.5% polyethylene glycol by mass percentage, 3 mg / mL proteinase K, and 0.1% ...
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